Quantitative Immunofluorescence to Measure Global Localized Translation

摘要

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

摘要

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

引言

The regulation of translation by different cellular functions has prompted many research teams to develop new tools and methods to determine the subcellular localization of mRNA translation and regulated protein synthesis^1,2,3,4. These recent technological advances allow for an improved understanding of the mechanisms involving translation upregulation or the repression of specific mRNAs during biological processes, such as neuronal development, drug response, and metastasis^5,6,....

方案

1. Determination of Puromycilation Conditions

NOTE: This technique describes the method used to assess localized translation during the MRC-5 cell adhesion process⁵. As puromycilation can be done in any cell, it is important to optimize the puromycilation conditions for the specific cell lines to be used, because the treatment conditions are not identical for each cell line in terms of the puromycin concentration and the desired incubation time. To show how these conditions are defined, three example cell lines (i.e., HeLa, MRC-5, and Huh-7) were treated with increasing concentrations of puromycin for differen....

结果

To accurately observe translation events using puromycin incorporation, it is critical to determine the optimal conditions for each cell line because each shows different puromycin incorporation kinetics (Figure 1)^9,11,12,18. Hence, to validate puromycin incorporation, it is necessary to treat the desired cell line with a standardiz.......

讨论

Recent technological advances have allowed for a better understanding of the mechanisms involved in translational upregulation or the repression of specific mRNAs in biological processes, such as neuronal development, drug response, and metastasis. The cost-effective methodology described here allows translation events to be visualized in cells to study how RNA-binding proteins regulate metastatic processes, such as cellular adhesion, migration, and invasion.

Although numerous methods to asses.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
DMEM	wisent	319-005-CL
胰蛋白酶	wisent	325-043 EL
FBS	Thermo Fisher Scientific	12483020
Puroycin 抗体 12D10	EMD millipore	MABE343	western blot 稀释度 1：25,000 免疫荧光稀释度 1：10,000
抗小鼠 IgG、HRP 偶联抗体	细胞信号传导技术	7076	Western Blot 稀释度 1：8,000
Western Lightning Plus-ECL	Perkin Elmer	NEL104001EA
抗小鼠 IgG （H+L）、F（ab'）2 片段（Alexa Fluor 488 偶联物）	细胞信号传输技术	4408	免疫荧光分离稀释度 1：400
CF568 鬼笔环肽	生物	00044	免疫荧光分离稀释度 1：400
环己胺	Sigma	C1988-1G	50µg/ml 终浓度
DAPI（4'，6-二脒基-2-苯吲哚，二盐酸盐）	Invitrogen	D1306	终浓度 1µg/ml
嘌呤霉素	生物碱性	PJ593	2.5&微量;g/ml 至 10µg/ml
Ibidi µ-培养皿 35 mm，高，ibiTreat	Ibidi	81156
MRC-5 细胞	ATCC	CCL-171
HeLa 细胞	ATCC	CCL-2
Huh-7 细胞	来自 Mazroui 博士（UniversitéLaval）
Fv1000	奥林巴斯		共聚焦成像系统
斐济软件			http://fiji.sc
PBS（磷酸盐水）	生物碱性	PD8117
甲醛 37% 溶液	生物碱性	C5300-1
Triton X-100	生物碱性	TB0198
BSA	Fisher 生物试剂	BP9702-100Tween20
	Fisher 生物试剂	BP337-500